Interferon was discovered by Isaacs and Lindenmann (Proc. R Soc. Lond[Biol.], 1957, 147, 258-267) in 1957 and has been known to have strong anti-viral effects.
Interferon is classified into type I interferon, including interferon-alpha/-beta, and type II interferon, including interferon gamma. Interferon-alpha is derived from either B lymphocytes or macrophages, interferon-beta is derived from fibroblasts and interferon-gamma is derived from T lymphocytes.
In human, at least 20 kinds of interferon-alpha genes and pseudo-genes have been identified. Proteins of these interferon-alphas are shown to have two disulfide bonds (Cys1-Cys98; Cys29-Cys138) in common. Human interferon-alpha does not contain an N-type glycosylation site but wild type mature protein contains an O-type glycosylation site at Thr106 (Adolf et al., Biochem. J., 276 (Pt 2), 511-518, 1991).
Interferon-alpha can be produced in cells of many tissues, however the yield is very low. Generally, it is produced largely in leucocytes such as monocyte/macrophage and B lymphocyte. Here, the proportion of subtypes of interferons produced depends on the cell type and production conditions. It has been known that the production of interferons is induced by virus infection. Further, bacteria, mycoplasma, protozoa and the like may induce the production of interferon and particularly, lipopolysaccharide (LPS) of gram negative bacteria is a strong interferon inducing agent.
Interferon-alpha mRNA is continuously produced even in tissues of a normal human (Tovey et al., Proc Natl Acad Sci USA, 1987, vol. 84, 5038-5042). It is believed that this interferon is an autocrine interferon playing a important role in cell growth and differentiation.
The working mechanism in vivo of interferon is not known yet. According to the report by Branca and Baglioni (Nature, 294, 768-770, 1981), it was shown that interferon-alpha and -beta bind the same receptor in human lymphoblastoid cell.
When virus infection takes place in vivo, interferon is produced and the produced interferon induced proteins, which perform interferon's functions. Representative examples of such induced proteins include 2′-5′-oligoadenylate synthetase and protein kinase phosphorylation of eIF2 (elongation factor2) which is a factor involved in initiation of peptide chain synthesis. The two enzymes are activated by double-stranded RNA (Lengyel P., Annu. Rev. Biochem., 51, 251-282, 1982; PestKa et. al., Annu. Rev. Biochem., 56, 727-777, 1982; De Maeyer and De Maeyer-Guignard J., interferons and other regulatory cytokines, Wiley, New York).
Interferon is clinically applied to treat chronic active hepatitis B, acute viral encephalitides, nasopharyngeal carcinoma and the like.
Since most of bioactive proteins used as medicaments show low stability in living bodies, patients who need bioactive proteins frequently receive excessive amounts to attain a therapeutic level of the proteins in the body. As a result, some patients suffer pain and inconvenience. Therefore, bioactive proteins having improved in vivo stability are desirable to alleviate the suffering of these patients.
International Patent Application Publication No. WO 98/48840 discloses a preparation of interferon alpha conjugated with polyethyleneglycol as a polymer to increase in vitro stability of bioactive proteins, while U.S. Pat. No. 6,399,103 discloses a preparation of a medicament by microcapsulation of human growth hormone. However, these methods require complicated processes involving primary production of a protein from a microorganism, followed by purification, and subsequent addition reactions. Also, cross-linking may take place at an undesired site leading to problematic heterogeneity of the final product. Another approach is a method using glycosylation.
Cell surface proteins and secretion proteins produced by eukaryotic cells can be modified by glycosylation. It is known that the glycosylation can affect not only physical properties of a protein but also stability and functions of a protein in living bodies.